Stable S-adenosylmethionine salts, the process for their preparation, and therapeutic compositions which contain them as active principle

ABSTRACT

New S-adenosylmethionine salts have been prepared which are stable indefinitely with time, even at elevated temperatures. 
     The new salts correspond to the formula: ##STR1## in which X is the equivalent of an organic sulphonic acid of pK less than 2.5, 
     and possess therapeutic activity in numerous fields of human therapy.

This invention relates to a new class of S-adenosylmethionine (SAM)salts which are highly stable at elevated temperatures and forpractically indefinite time periods.

It is known that S-adenosylmethionine is a product of natural originpresent in all living organisms, in which it is actively synthesised bya specific enzyme, and corresponds to the following structural formula:##STR2## SAM participates in a great number of metabolic processes offundamental importance for the human organism, and consequently itsdeficiency lies at the basis of many organic malfunctions.

Although the biological importance of this product has been known forsome decades, the possibility of testing it and thus using it as a drughas existed only in recent years, because of its extreme instability attemperatures exceeding 0° C.

In this respect, only in 1975 did the present applicant succeed inpreparing a SAM salt which was sufficiently stable at 25° C. (U.S. Pat.No. 3,893,999), followed by some salts having good stability at 45° C.(U.S. Pat. No. 3,954,726/1976 and U.S. Pat. No. 4,057,686/1977).

More specifically, U.S. Pat. No. 3,893,999/1975 describes SAMtriparatoluene sulphonate, U.S. Pat. No. 3,954,726/1976 describes SAMdisulphatediparatoleune sulphonate, and U.S. Pat. No. 4,057,686/1977describes a group of SAM salts which can be indicated overall asSAM.4RSO₃ H or SAM.3RSO₃ H in which RSO₃ H indicates a sulphonic acidequivalent which can be partly substituted by the equivalent ofsulphuric acid.

The said applicant has stated that he is unable to explain the reasonwhy only the specific claimed salts were stable, when SAM salts preparedunder the preceding state of the art (namely the monochloride--U.S. Pat.No. 2,969,353--and disulphate--West German patent application No.1,803,978) had at the most attained a limited stability with time ifmaintained at 4° C., nor why triparatoluene sulphonate was stable onlyup to 25° C., while disulphatediparatoluene sulphonate was stable up to45° C.

It has now been quite unexpectedly found, and forms the subject matterof the present invention, that stable SAM salts are obtained wheneverSAM is salified with 5 moles of an organic sulphonic acid of pK lessthan 2.5.

More specifically, it has been surprisingly found that if SAM issalified with 5 moles of an organic sulphonic acid of pK less than 2.5,a salt of maximum stability is obtained.

Salts containing 4 or 6 moles of acid have a stability which is stillgood, but decidedly less. Salts containing 1 to 3 moles of acid areabsolutely unacceptable for therapeutic use in that they are subject tovast degradation phenomena.

It must be emphasised that as the new salts according to the inventionall find application in human therapy, the presence of even a smallpercentage of a degraded product is unacceptable not only because itimplies a corresponding loss of activity, but also and in particularbecause it indicates the formation of metabolites which have been foundto be slightly toxic and have the capacity to interfere with biologicalprocesses.

It has also been found, and represents a further subject matter of thepresent invention, that the stability of the new SAM salts is directlyinfluenced by the polarity of the environment, and thus in particular bythe quantity of moisture present, and means have therefore been found toreduce this moisture to values close to zero.

The salts according to the present invention correspond to the generalformula ##STR3## in which X is the equivalent of an organic sulphonicacid of pK less than 2.5.

In particular, it has been found that salts of optimum stability areobtained when X is CH₃ --SO₃ ⁻ or CH₃ --C₆ H₄ --SO₃ ⁻. The pK of thesetwo acids is as follows:

methanesulphonic acid: pK<0.5

p.toluenesulphonic acid: pK<0.5

The new salts have proved very useful in numerous fields of humantherapy, for example as hepato-protectors as will be more apparenthereinafter.

They are prepared by a process comprising the following essentialstages, which are all critical for the purpose of obtaining a product ofabsolutely constant and reproducible pharmaceutical purity:

(a) preparing a concentrated aqueous solution of a crude SAM salt by anyknown method;

(b) purifying the solution by chromatography, by passage through aweakly acid ion exchange resin column;

(c) eluting the SAM with a dilute aqueous solution of the required acid;

(d) titrating the eluate and adjusting the acid quantity to the strictlystoichiometric proportion relative to the SAM present;

(e) concentrating the eluate;

(f) lyophilisation.

The aqueous solution prepared in stage (a) can obviously contain anysoluble SAM salt because the anion is eliminated in the next passagethrough the column, and therefore does not interfere with the rest ofthe process. In general, with the normal processes involvingconcentration and extraction of SAM from yeast, a solution is obtainedcontaining the SAM⁺ ion and the SO₄ ⁻⁻ ion.

In all cases, the pH of the solution is adjusted to between 6 and 7, andpreferably 6.5.

The chromatographic purification stage (b) is carried out preferablywith Amberlite IRC50 or Amberlite CG50.

The elution of stage (c) is preferably carried out with a 0.1 N aqueoussolution of the required acid.

If titration of the eluate (stage d) shows that the quantity of acidequivalents present is less than 5, this being the usual case, then thatquantity of acid corresponding exactly to the deficiency is added in theform of a concentrated commercial aqueous solution. However, if it isshown that an excess of acid is present, this is eliminated by treatingthe solution with strong basic ion exchange resin in OH⁻ form, forexample Amberlite IRA-401.

In stage (e), the eluate is concentrated to an optimum value for thesubsequent lyophilisation process, i.e. to a value of between 50 and 100g/l, and preferably around 70 g/l.

The final lyophilisation is carried out by the usual methods, to give aperfectly crystalline salt of 100% purity.

If lyophilisation is carried out in the presence of a suitable inertsubstance, a product is obtained having a smaller residual moisturecontent, and thus more stable.

More specifically, it has been found that if the prepared salt isintended for use in injectable pharmaceutical forms, lyophilisationshould be carried out in the presence of mannitol. If however the newsalt is intended for the preparation of oral tablets, lyophilisationshould be carried out in the presence of powdered silicic acid.

Some practical preparation examples are given hereinafter for purelyillustrative purposes, in order to make the new products more easilyreproducible.

EXAMPLE 1

110 l of ethyl acetate and 110 l of water are added at ambienttemperature to 900 kg of yeast enriched with SAM (6.88 g/kg) inaccordance with Schlenk (Enzymologia, 29, 283 (1965)).

After energetically stirring for 30 minutes, 500 l of 0.35 N sulphuricacid are added, and stirring is continued for a further one and a halfhours.

The mixture is filtered and the residue washed with water to give 1400 lof solution containing 4.40 g/l of SAM, equivalent to 99.5% of thatpresent in the starting material.

A solution of 11.5 kg of picrolonic acid in 120 l of n-butanol is addedto 700 liters of this solution under stirring.

After leaving overnight, the precipitate is separated by centrifugingand washed with water.

The precipitate is dissolved by stirring it at ambient temperature into31 liters of a 1 N solution of sulphuric acid in methanol.

250 l of benzene are then added.

After the precipitate has completely sedemented, the supernatantsolution is decanted, and the insoluble residue is washed with a littleether.

The precipitate is dissolved in 400 l of distilled water, 1 kg ofdecolourising charcoal are added, and the mixture filtered.

A column of Amberlite IRC 50 resin in H⁺ form is prepared and carefullywashed with distilled water.

4.8 kg of glacial acetic acid are added under stirring to the previouslyobtained aqueous solution, and 2 N NaOH is then added until a pH of 6.5is obtained.

The solution is passed through the resin column at a rate of 400 l/h,which is maintained constant during the entire process.

200 l of distilled water, 1600 l of 0.1 M acetic acid and a further 200l of distilled water are then passed through successively.

The SAM from the ion exchange column is eluted with 200 l of a 0.1 Nsolution of methanesulphonic acid.

The eluate is concentrated under vacuum to 30 l, after which asufficient quantity of methanesulphonic acid is added until a molarmethanesulphonic acid/SAM ratio of 5:1 is obtained, and the solution isthen lyophilised.

4 kg of product are obtained having the following composition: SAM⁺44.7%; CH₃ SO₃ H 53.8%; H₂ O 1.5%.

The product is soluble to the extent of more than 20% in water, but islittle soluble in common organic solvents.

Thin layer chromatography shows that the compound is free from anyimpurity.

The analytical data are as follows:

    ______________________________________                                        empirical formula: C.sub.20 H.sub.42 N.sub.6 O.sub.20 S.sub.6.0.5H.sub.2                  N %      S %    SAM %                                             ______________________________________                                        calculated    9.47       21.65  44.9                                          found         9.41       21.68  44.7                                          E 1% 256 nm = 164                                                             (6N H.sub.2 SO.sub.4).                                                        ______________________________________                                    

On repeating the process in an identical manner, but usingp-toluenesulphonic acid for the column elution, it is possible to obtaina salt of formula: ##STR4## of which the analytical data are as follows:

    ______________________________________                                        empirical formula: C.sub.50 H.sub.62 N.sub.6 O.sub.20 S.sub.6.0.5H.sub.2                  N %      S %    SAM %                                             ______________________________________                                        calculated    6.63       15.15  31.4                                          found         6.81       15.43  31.5                                          E 1% 256 nm = 115                                                             (6N H.sub.2 SO.sub.4).                                                        ______________________________________                                    

EXAMPLE 2

The procedure described in Example 1 is followed exactly as far as thecolumn elution.

The SAM is eluted with an aqueous 0.5 N methanesulphonic acid/0.5 Nsulphuric acid solution.

After concentrating under vacuum and filtering with activated carbon,the solution is titrated and the two acids are added in an exactlymeasured quantity to obtain the salt SAM.1.5H₂ SO₄.2CH₃ SO₃ H.0.5H₂ O.

This salt is obtained with pharmaceutical purity by lyophilisation ofthe solution.

Its analytical data are as follows:

    ______________________________________                                        empirical formula: C.sub.17 H.sub.33 N.sub.6 O.sub.17 S.sub.4.5.0.5H.sub.2     O                                                                                        N %      S %    SAM %                                             ______________________________________                                        calculated    11.26      19.30  53.4                                          found         11.13      18.98  53.3                                          E 1% 256 nm = 195                                                             (6N H.sub.2 SO.sub.4)                                                         ______________________________________                                    

All possible mixed salts can obviously be prepared by varying the ratioof the two acids.

On repeating the process in an identical manner but usingp.toluenesulphonic acid/sulphuric acid mixtures for the elution, thefollowing double salt is obtained: SAM.1.5H₂ SO₄.2CH₃ --C₆ H₄ --SO₃H.0.5H₂ O of which the analytical data are as follows:

    ______________________________________                                        empirical formula: C.sub.29 H.sub.41 N.sub.6 O.sub.17 S.sub.4.5.5H.sub.2                  N %      S %    SAM %                                             ______________________________________                                        calculated    9.35       16.04  44.3                                          found         9.27       15.95  44.1                                          E 1% 256 nm = 162                                                             (6N H.sub.2 SO.sub.4)                                                         ______________________________________                                    

It is also possible to obtain an entire series of mixed salts in thiscase, by varying the ratio of the two added acids.

EXAMPLE 3

The preparation described in example 1 was repeated identically, but2.50 kg of apyrogenic mannitol were added to the solution beforelyophilisation.

The solution was then lyophilised in the usual manner.

The addition of mannitol as a lyophilisation support enables a productto be obtained having a residual moisture content of 0.1%.

The product obtained in this manner is suitable for conversion intoinjectable pharmaceutical forms.

EXAMPLE 4

The procedure of Example 1 is followed. 2 kg of Aerosil (pulverisedsilic acid) are added to the solution before lyophilisation, and theresultant colloidal suspension is lyophilised.

The addition of Aerosil as a lyophilisation support enables a product tobe obtained having a residual moisture content of 0.2%.

The product obtained in this manner is suitable for conversion intotablets for oral use.

Stability tests were carried out at 45° C. on the salts prepared in thedescribed manner, by determining the percentage of residual salt ofbasic SAM⁺ at fixed times.

The residual SAM percentage at the times indicated was determined by thenew method described hereinafter, which ensures maximum accuracy ofmeasurements in that it enables the SAM to be totally separated from allpossible degradation products.

In this respect, it has been found that with the method used up to thepresent time, based on an analytical column of Dowex 50 ion exchangeresin (Schlenk and De Palma: J. Biol. Chem. 229 (1957)), the separationof the SAM from certain degradation products, and in particular frommethylthioadenosine, was not complete and therefore led to an error inevaluating the stability of the SAM, which appeared better than it was.

The present method for determining the SAM is based on the use of HPLC.

Analytical conditions used:

column: PARTISIL 10 SXC-2.5×250 mm

eluent: 0.1 M ammonium formate of pH 4, containing 20% of methanol forHPLC

flow: 1 ml/minute

SAM retention time: about 400 seconds.

                  TABLE 1                                                         ______________________________________                                        SAM.n CH.sub.3 SO.sub.3 H                                                             DEGRADATION AT 45° C. AFTER                                         residual 60       120   180    240   360                                 n    moisture days     days  days   days  days                                ______________________________________                                        1    0.2%     100%     100%  100%   100%  100%                                2    0.5%     80%      100%  100%   100%  100%                                3    0.8%     30%       60%  80%    100%  100%                                4      1%      5%       10%  14%     17%   20%                                5    1.5%     --        1.5%  3%     4.5%  5%                                 6      2%      5%       10%  14%     17%   20%                                ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                         ##STR5##                                                                               DEGRADATION AT 45° C. AFTER                                       residual 60       120   180    240   360                                 n    moisture days     days  days   days  days                                ______________________________________                                        1    0.2%     100%     100%  100%   100%  100%                                2    0.5%     80%      100%  100%   100%  100%                                3    0.8%     30%       60%  80%    100%  100%                                4      1%      5%       10%  14%     17%   20%                                5    1.5%     --        1.5%  3%     4.5%  5%                                 6      2%      5%       10%  14%     17%   20%                                ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        SAM.1.5 H.sub.2 SO.sub.4.n CH.sub.3 SO.sub.3 H                                        DEGRADATION AT 45° C. AFTER                                         residual 60       120   180    240   360                                 n    moisture days     days  days   days  days                                ______________________________________                                        1    1%       5%       10%   14%    17%   20%                                 2    1.5      --        1.5%  3%     4.5%  5%                                 3    2%       5%       10%   14%    17%   20%                                 ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                         ##STR6##                                                                               DEGRADATION AT 45° C. AFTER                                       residual 60       120   180    240   360                                 n    moisture days     days  days   days  days                                ______________________________________                                        1    1%       5%       10%   14%    17%   20%                                 2    1.5%     --        1.5%  3%     4.5%  5%                                 3    2%       5%       10%   14%    17%   20%                                 ______________________________________                                    

From the data of tables 1, 2, 3 and 4 it is apparent that, completelyunforeseeably, SAM salts attain maximum stability when they contain 5acid equivalents. Salts with 4 or 6 equivalents still have goodstability, whereas salts with a lower number of acid equivalents have nopractical use because of their instability.

It is also apparent that the stability takes the same patternindependently of the acid used, provided that it is an organic sulphonicacid of pK<2.5.

It was not possible to prepare salts with acids of pK>2.5 containing 4to 6 acid equivalents.

Stability tests were also carried out by the same method on saltsprepared in the presence of a lyophilisation support, in accordance withexamples 3 and 4.

The results of these determinations are given in the following tables 5and 6. The drastic reduction in moisture content when lyophilisation iscarried out in the presence of a support is apparent, together with theconsequent total stability of the salts which is attained in thismanner.

                  TABLE 5                                                         ______________________________________                                        SAM.5CH.sub.3 SO.sub.3 H                                                                 RESIDUAL DEGRADATION AT                                                       MOISTURE 45° C. AFTER 360 DAYS                              ______________________________________                                        Lyophilised  1.5%       5%                                                    without support                                                               Lyophilised with                                                                           0.1%       --                                                    mannitol                                                                      (100 g SAM.sup.+  + 120 g                                                     mannitol)                                                                     Lyophilised with                                                                           0.2%       --                                                    Aerosil                                                                       (100 g SAM.sup.+  + 100 g                                                     Aerosil)                                                                      ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        SAM.5CH.sub.3 --C.sub.6 H.sub.4 --SO.sub.3 H                                             RESIDUAL DEGRADATION AT                                                       MOISTURE 45° C. AFTER 360 DAYS                              ______________________________________                                        Lyophilised  1.5%       5%                                                    without support                                                               Lyophilised with                                                                           0.1%       --                                                    mannitol                                                                      (100 g SAM.sup.+  + 120 g                                                     mannitol)                                                                     Lyophillised with                                                                          0.2%       --                                                    Aerosil                                                                       (100 g SAM.sup.+  + 100 g                                                     Aerosil)                                                                      ______________________________________                                    

The salts SAM.5CH₃ SO₃ H and SAM.5CH₃ --C₆ H₄ --SO₃ H were tested in awide pharmacological screening trial, and in all cases showed highlyinteresting activity and toxicity characteristics which were independentof the anion bonded to the SAM. It was established that the activity ofthe new salts depends substantially on the capacity of the SAM⁺ ionreleased in the organism to act as a donor of methyl groups, as thenatural substrate of a large number of transmethylase enzymes whichcatalyse fundamental reactions of the lipid, protide and glucidemetabolism.

The importance of the new salts thus derives substantially from the factthat they make the S-adenosylmethionine absolutely stable attemperatures up to 45° C., so enabling its transmethylating activity inthe human organism to be utilised 100% without the risk of formation oftoxic degradation products which interfere negatively with thebiological processes activated by the SAM⁺.

Toxicity

The acute toxicity in the mouse was determined, the following valuesbeing obtained for both salts:

LD₅₀ by oral administration >3 g/kg

LD₅₀ by intravenous administration 1.1 g/kg

Tolerability and chronic toxicity tests were carried out on rats of theWistar and Sprague-Dowley stock, by administering 20 mg/kg per day ofproduct for 12 months. On termination of the treatment, the variousorgans and systems showed no pathological alteration.

Teratogenesis tests were carried out on rabbits. When salt doses tentimes greater than the maximum therapeutic doses were administered, noteratogenic action or malformative action on the embryos or terminalfetuses was encountered.

Intravenous administration of doses up to 200 mg/kg caused no pyrogenicmanifestation in the rabbit.

Venous administration of 40 mg/kg in the rabbit and rat caused no changein carotid pressure, cardiac and respiratory frequency, or in theelectrocardiograph trace.

Local tolerability of intramuscular injection, even afteradministrations repeated for 30-60 days, and of intravenous injection inthe marginal vein of the outer ear of the rabbit, was excellent.

Pharmacology

An entire series of tests carried out on rats have shown that the newsalts exert a very considerable protective and resolving action inhepatic steatosis induced by a hyperlipid-hyperprotein diet inaccordance with Handler, and in steatosis induced by acute alcoholicintoxication and other toxic agents even when administering doses of 10mg/kg of SAM⁺.

In experimental hyperlipemia in the rat, for example induced by TritonS, the new salts have demonstrated a very conspicuous hypolipemicactivity which, in relation to the dose used, i.e. 10 mg/kg (againexpressed in SAM⁺), was much more intense than in the case of otherdrugs of hypolipemic activity.

In chickens rendered atherosclerotic by means of diets enriched incholesterol and fructose, parenteral administration of the new productin doses of 10 mg/kg reduced the cholesterolemia and favourably modifiedlesions encountered in the controls with respect to the thoracic andabdominal aorta and the small vessels of the encephalic base.

With regard to phospholipid metabolism, it was found experimentally thatthere was an increase in the phosphatidylcholine quantity in the hepatictissue of rats with uncompensated steatosis. A clear increment in thephosphatidylcholine was also determined at the expense of the hematicα-lipoproteins in experimental alterations caused by β/α lipoproteinratios.

All these tests have clearly indicated a curative effect of the newsalts in alterations of the lipid metabolism.

A further series of tests carried out on the rat have shown thatadministration of 1 mg/kg doses induces an accumulation of glycogenicreserves at the hepatic and muscular level, which is demonstrated bothby histochemical methods and by quantitative determinations. Inexperimental diabetes induced by alloxan, the insulin quantity necessaryfor returning glycemia values to normal was considerably reduced byadministrations equivalent to 0.5 mg/kg of SAM⁺.

This series of tests has demonstrated a clear positive action of the newcompounds according to the invention on the glucide metabolism.

Finally, rats with experimentally induced hypodisproteinemia weretreated with quantities of 10 mg/kg of SAM. It was found that saidproduct returns the total proteinemia values to normal, by substantiallyincreasing the albumin level and thus showing marked protein anabolicactivity.

This and other similar tests have demonstrated the curative power of thenew products in malfunctions of the protide metabolism.

Summarising, on the basis of the aforesaid pharmacological tests and ofmany others which have enabled the activity of the new salts to beexplored at all levels in the human organism, the activity of the newproducts has been clinically established in hepatology in the case ofacute and chronic hepatic intoxication, in neurology as anantidepressive, and in osteology in the case of rheumatoid arthritis.

The activity in numerous other fields of human therapy is underinvestigation.

The new salts can be administered orally, or by intramuscular orintravenous injection.

Other possible administration forms are suppositories, liquids forocular installation, aerosol, or forms for topical application.

I claim:
 1. S-adenosylmethionine (SAM) salts of formula: ##STR7## inwhich X is the acid equivalent of an organic sulphonic acid of pK lessthan 2.5.
 2. A composition for the treatment of hyperlipemia comprisingan anti-hyperlipemic effective amount of the compound of claim 1 and atherapeutically acceptable carrier.